The present invention relates to transgenic cell populations, e.g., transgenic cell lines and transgenic animals. More particularly, the invention relates to negative selection methods, chimeric gene constructs useful therefor, methods for establishing stable transgenic cell populations and then selectively ablating (i.e., negatively selecting for) specific cell types and/or cell lineages in such transgenic cell populations at desired stages of development or differentiation.
Positive selection procedures or negative selection procedures can be used to differentiate various cell types of a given cell population. In a positive selection situation, cells having desirable properties have one or more growth and/or maintenance advantages over cells which do not have such properties. The cells having such desirable properties out-compete the remaining cells in the cell population, and eventually overtake the remaining cells in the population. Thus, the ultimate cell population is determined, in large part, by specific features possessed by the surviving cells (but which features were not possessed by the cells which were eliminated).
In contrast, negative selection involves removing from a cell population (or destroying) those cells which do not have the specific features desired for the ultimate cell population. Thus, in the negative selection situation, the ultimate cell population is determined by one or more specific features possessed by the cells which are no longer a part of the surviving cell population.
While positive selection techniques have found widespread applicability in the field of biology, negative selection techniques have been less widely used.
The ability to more broadly apply negative selection techniques to biological systems would make possible a variety of therapeutic, prophylactic and research applications of such techniques.
For example, inadequate selectivity currently severely limits the potential efficacy of most cancer chemotherapy. Inadequate selectivity also limits the therapeutic use of many traditional chemicals, due to the likelihood of undesirable side-effects upon administration of the therapeutic agent in dose levels effective to impart the desired therapeutic effect.
Since most chemotherapeutic agents target the proliferative functions in neoplastic cells, the dosage levels of such agents must generally be limited to suboptimal levels to avoid unacceptable toxicity to normal stem cells, whose proliferation sustains vital hematopoietic and epithelial tissues. It would, therefore, be desirable to be able to selectively direct the effects of the therapeutic agent to the affected cell population, and not to the total cell population indiscriminately.
Another area where it would be desirable to be able to selectively direct the effects of treating agents is in the study of critical events in development and differentiation. For example, a major problem in the study of vertebrate development is the absence of a genetic approach for the study of such critical events in cell lineage development and differentiation. Various attempts have been made, using both mechanical and chemical methods, to specifically eliminate one cell type in a living organism. Although some success has been achieved, the complex structure of the vertebrate system has not allowed selective destruction of a single cell type to be accomplished employing prior art methods.
Recently, Palmiter, et al., in Cell, 50:435-443 (1987) demonstrated that transgenic mice lacking normal pancreas function could be created when fertilized mouse eggs were microinjected with a vector construct in which the elastase I promoter/enhancer was fused to a gene coding for a toxic gene product (the diphtheria toxin A polypeptide). These investigators report that some of the transgenic mice had no pancreas, while others had a small rudimentary organ which resembled an embryonic pancreas. Based on these observations, the authors suggest that the difference in development of the transgenic pancreas probably reflects differential timing of expression of the diphtheria toxin A polypeptide, in individual mice.
Breitman, et al., in Science, 238:1563-1565 (1987), have also demonstrated that the mouse gamma 2-crystallin promoter can be used to direct expression of the toxic gene product, diphtheria toxin A, in eye lens tissues in transgenic mice. Thus, Breitman et al. report the generation of a line of mice in which embryonic fiber cells within the lens have been genetically ablated.
Unfortunately, one problem with genetic ablation of a cell line during embryonic life is that expression of some toxin genes may be lethal to the early embryo or sufficiently toxic to prevent establishment of a stable transgenic pedigree. In addition, in order to study cell lineages, e.g., cells of the immune system, it would be useful to be able to control not only the timing, but also the degree of cellular ablation.
The ability to regulate cell ablation would make possible (1) the development of animal models mimicking various disease states, (2) treatment of various disease states, and (3) the controlled elimination from a cell population (e.g., a subject) of a cell line capable of providing to the subject a desirable component (as an exogenous gene product), once the need for such a component no longer exists, or once the subject achieves the ability to produce sufficient quantities of such component as an endogenous product.
There is, therefore a need for improved genetic ablation methods (i.e., negative selection methods) that will allow controlled manifestation of a toxic phenotype in transgenic cell populations. An improved negative selection (i.e., genetic ablation) method should permit the generation of transgenic cell populations, and it would preferably be inducible, so that the treatment of the transgenic cell population can be carried out when desired, e.g., for an intact organism, when such organism is in its embryonic and developing states, or when it is mature. Furthermore, the improved method should allow the extent or degree of the cellular ablation to be controlled. Finally, the improved method should make it possible to study the capacity a specific cell type has for regeneration. This could be done by ablating a specific cell type by treating the subject for a finite period of time, withdrawing the drug treatment, and then examining the plasticity or repopulation of the cell type system over time.
To fulfill the need for an improved negative selection (i.e., selective cell) method, the present invention discloses a new method for selectively, ablating, by genetic means, specific cell lineages in transgenic cell populations. The new negative selection method, which permits the production of stable transgenic cell populations, and thus can be used at various stages of development of a transgenic cell population, also allows both the onset and degree of cellular ablation to be controlled. Such control not only allows specific types of cells to be studied, but also enables the examination of the capacity of residual stem cells for regeneration.
Thus, the method of the invention is extremely useful, for example, for the study of immunological and neuroendocrine disorders, and possibly neurological disorders; in addition to being useful for the treatment of various disorders; and potentially useful for selectively eliminating cell line(s) which at one time served a useful purpose, but which is no longer required for normal functioning of the host organism.